Nuclear magnetic resonance (NMR) and other spectroscopic studies are planned to probe the structure and reactivity of proteins (peroxidases, cytochrome P450, heme oxygenase) and model metal tetrapyrrole complexes involved in the metabolism and activation of dioxygen, hydroperoxides, and related small molecules. The paramagnetic nature of most of the active forms of these proteins and their synthetic models "lights up" the active site with an expanded scale of hyperfine shifts that enhances resolution, allows complex, multicomponent reacting systems to be analyzed, and provides unique information on the magnetic properties and electronic structure of these chromophores. Promising preliminary advances in the application of multi-pulse 2D NMR methodology to paramagnetic tetrapyrrole complexes and proteins will be thoroughly developed for resonance assignment and electronic/magnetic/molecular structure determination on both models and proteins. Emphasis will be given to problems posed by paramagnetic enzymes that are considered large by conventional NMR standards. These techniques will be employed to characterize the identity, disposition, and protonation state of key residues within the heme pocket, the catalytically relevant hydrogen-bonding networks, and the sites of substrate binding in a variety of plant, fungal, and mammalian heme peroxidases. They will also be used to probe the influence of substrate binding on the structure and dynamic properties of these active sites. The scope and limitations of the 2D methodology for bacterial heme mono- oxygenases will also be explored. Skeletally modified porphyrins (chlorins, isobacteriochlorins, substituted) are important as prosthetic groups in some proteins (nitrate, sulfate reductases), while other skeletally modified porphyrins (N-substituted porphyrins, oxophlorins) are formed purposefully (heme oxygenase) or accidently (cytochrome P45) in other metabolic cycles, most of which involve dioxygen utilization. Application of the new 2D methodology will be used to probe the electronic structure of these modified porphyrins, to understand the functional consequences of the skeletal modification, and to facilitate identification of these altered forms in proteins and in reactive model systems. Detailed studies of the coordination chemistry of important but poorly characterized iron complexes of isoporphyrins and oxophlorins will provide the basis for understanding their role in heme degradation or modification. Studies of reactive intermediates allow identification of functional forms involved in these oxidative processes. These studies will focus on models for the reactivity of coordinated alkyl peroxides, the mechanism of dioxygen "insertion" into metal-carbon bonds (to form peroxide ligands), the mechanisms of oxophlorin formation and destruction, and in proteins, the identification of isoporphyrins and the site of radical delocalization in HRP-Compound I.